Stem cells (sc) are almost certainly the cells of origin of intestinal cancer. But, sc mechanisms are obscure because there are no specific markers to identify SO. Our long-term objective is to use animal models to develop & test research tools for identifying adult intestinal sc in vivo. Here we will test one tool, GFP-Tcf4 transgenic mice. We designed this mouse line to display green fluorescence protein positive (GFP+) cells when Tcf4 is activated. Tcf4 is important because 1) intestinal tumors have activated Tcf4 due to cancer-causing Apc mutations & 2) available evidence suggests that Tcf4 activation in normal crypts is unique to sc. Thus, a marker for Tcf4 activation may be a marker for sc. We crossed GFP-Tcf4 mice with ApcMin mice because the latter are at high risk for intestinal cancer & to assess effects of Apc mutations on Tcf4 activation. AIM 1: To identify (ID), isolate & characterize GFP+ enterocytes from GFP-Tcf4 mice. GFP+ enterocytes in these mice appear localized to the crypt base (where SC reside). To quantify this finding, we will a) use fluorescence microscopy/advanced image analysis (of intestinal sections) measuring in vivo GFP signal intensity along the crypt axis, & b) flow cytometry sorting (of isolated fixed, GFP+ enterocytes derived from purified whole crypts). Hypothesis 1(H1): GFP+ enterocytes are only in the crypt base, are a small % of crypt cells, stain for nuclear (-catenin ((c). AIM 2. To see, using microscopy & cytometry as above, if the GFP reporter responds to changes in Tcf4 activation. H2: The # of crypt cells that are GFP+ & show nuclear (c localization are increased in mutant crypts (normal-appearing mucosa [1 Apc mutation], adenomatous mucosa [2 mutations]). AIM 3. To isolate viable GFP+ & GFP- enterocytes. Here we will isolate viable (not fixed) enterocytes & sort them by flow cytometry. H3: Prevention of clumping in single cell suspensions of enterocytes will permit flow cytometric sorting of viable cells; GFP+ but not GFP- cells will show nuclear (c staining & test positive for Hoechst dye efflux, a functional SC property. Significance. If predictions hold, Aim 1 results will suggest that GFP expression ID's intestinal SC (or sc-like cells); Aim 2 results, that the reporter responds to changes in Tcf4 activation; Aim 3 results, that viable GFP+ intestinal cells are isolatable and characterizable hence can be tested to see if they have other sc properties. One could then evaluate the role of SC in initiation & promotion of intestinal neoplasms.